Method of improving crop yields

ABSTRACT

The invention provides a method of improving the yield of a crop. The method comprises growing transgenic plants to produce a crop, the transgenic plants being able to metabolize at least one synthetic auxin. A synthetic auxin is applied to the plants at least once during their growth, the synthetic auxin being one that can be metabolized by the transgenic plants. Finally, the crop is harvested.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C.§119(e) from U.S. Provisional Application Serial No. 60/345,346, filedOct. 24, 2001. The entire disclosure of U.S. Provisional ApplicationSerial No. 60/345,346 is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to increasing crop yields. Inparticular, the present invention relates to increasing crop yields byapplying an auxin herbicide to transgenic plants capable of metabolizingthe applied auxin herbicide.

BACKGROUND OF THE INVENTION

[0003] 2,4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide usedprimarily to control dicotyledonous weeds. 2,4-D is broken down in soilby a variety of microorganisms, including Alcaligenes eutrophus. A gene(tfdA) has been isolated from strains of A. eutrophus which encodes thefirst enzyme in the 2,4-D degradation pathway of these bacteria. Thisenzyme is a dioxygenase which catalyzes the conversion of 2,4-D to2,4-dichlorophenol (DCP).

[0004] Transgenic tobacco plants, cotton plants, and hardwood treescontaining the tfdA gene have been reported to have increased toleranceto 2,4-D. Streber et al., Bio/Technology, 7, 811-816 (1989); Lyon etal., Plant Molec. Biol., 13, 533-540 (1989); Bayley et al., Theor. Appl.Genet., 83,645-649 (1992); Llewellyn and Last, in Herbicide-ResistantCrops Chapter 10, pages 159-174 (Duke, ed., CRC Lewis Publishers 1996);Last and Llewellyn, Weed Science, 47, 401-404 (1999); U.S. Pat. Nos.5,608,147, 6,100,446, and 6,153,401; PCT application WO 95/18862.However, 2,4-D-tolerant transgenic cotton has been reported to havesignificantly reduced growth rates when sprayed with 2,4-D at levelsthat might be encountered in agricultural situations. Last andLlewellyn, Weed Science, 47, 401-404 (1999).

[0005] Increases in growth and yields have been reported to occur fromapplications of sublethal concentrations of 2,4-D and other herbicidesto plants sensitive to them. See, Allender, J. Plant Nutrition.,20,69-80(1997); Moffett et al., Crop Sci., 20, 747-750(1980); Wiedmanand Appleby, Weed Res., 12, 65-74 (1972); Miller et al., Crop Sci., 2,114-116 (1962); McIlrath and Ergle, Botanical Gazette, 461-467 (1953);McIlrath and Ergle, Plant Physiol., 693-702 (1952). However, the growthstimulation is usually small, highly variable, transitory, and difficultto reproduce in the field. Allender et al., J. Plant Nutrition., 20,81-95 (1997).

SUMMARY OF THE INVENTION

[0006] The invention provides a method of improving the yield of a crop.The method comprises growing transgenic plants to produce a crop, thetransgenic plants being able to metabolize at least one synthetic auxin.A synthetic auxin is applied to the plants at least once during theirgrowth, the synthetic auxin being one that can be metabolized by thetransgenic plants. Finally, the crop is harvested.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007]FIG. 1: Diagram of pProPClSV-SAD.

[0008]FIG. 2: Diagram of pPZP21-PNPT-311g7.

[0009]FIG. 3: Diagram of pPZP21-PNPT-512g7.

[0010]FIG. 4: Diagram of pPZP21-PNPT-311-SAD.

[0011]FIG. 5: Diagram of pPZP21-PNPT-512-SAD.

[0012] In these figures, SAD=2,4-D-degrading synthetic gene adapted fordicots; CDS coding sequence; AMV-Leader=5′ untranslated leader sequencefrom the 35S transcript of alfalfa mosaic virus; PClSV-Promoter=peanutchlorotic streak virus promoter; T-Left =T-DNA left border fromAgrobacterium tumefaciens nopaline Ti plasmid pTiT37; 35SPolyA=3′polyadenylation (polyA) termination signal sequence from the cauliflowermosaic virus (CaMV) 35S transcript; NPTII=neomycin phosphotransferaseII; g7PolyA=3′ polyA termination signal from gene 7 within the T-Leftborder of an A. tumefaciens octopine plasmid; MCS=multiple cloning site;T-Right=T-DNA right border from A. tumefaciens Ti plasmid pTiT37.

DETAILED DESCRIPTION OF THE PRESENTLY-PREFERRED EMBODIMENTS OF THEINVENTION

[0013] “Synthetic auxins” are compounds generally used as herbicides.Thus, they are also referred to as “auxin herbicides.” Preferredsynthetic auxins (auxin herbicides) for use in the present invention arethe phenoxy auxins (phenoxy herbicides), which include4-chlorophenoxyacetic acid (4-CPA), 2,4-dichlorophenoxyacetic acid(2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA),2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4-dichlorophenoxybutyricacid (2,4-DB), 4-(2-methyl-4-chlorophenoxy)butyric acid,2-(4-chlorophenoxy)propionic acid, 2-(2,4-dichlorophenoxy)propionicacid, 2-(2,4,5-trichlorophenoxy)propionic acid, and salts and esters ofthese acids. Most preferred are 2,4-D, 2,4-DB, and esters thereof. Auxinherbicides, including phenoxy herbicides, are available commercially.See Crop Protection Reference (Chemical & Pharmaceutical Press, Inc.,New York, N.Y., 11th ed., 1995).

[0014] A transgenic plant according to the invention is a plant which istolerant to at least one synthetic auxin to which the correspondingnontransgenic plant is sensitive. “Tolerant” means that the transgenicplant can grow in the presence of an amount of an auxin herbicide whichinhibits the growth of the corresponding nontransgenic plant and/or thatthe transgenic plant is not injured by an amount of auxin herbicidewhich injures the nontransgenic plant. “Sensitive” means that thenontransgenic plant is injured or killed by one or more auxinherbicides. In particular, nontransgenic dicotyledonous plants areseverely injured or killed by auxin herbicides. Nontransgenicmonocotyledonous plants are much less sensitive to auxin herbicides thandicotyledonous plants, but monocotyledonous plants can be injured byauxin herbicides applied to them at particular developmental stages(e.g., grain fill) or during times of stress. The transgenic plants ofthe invention are tolerant because they are able to metabolize one ormore synthetic auxins as a result of the expression of heterologous DNAcoding for one or more enzymes which metabolize the synthetic auxin(s)so that the synthetic auxin(s) are no longer harmful to plants.“Heterologous DNA” is used herein to mean DNA not found in the plant,such as DNA from a microorganism or another species or strain of plant.

[0015] To prepare the transgenic plants of the invention, a DNA moleculecomprising DNA coding for an enzyme or enzymes which metabolize(s) atleast one synthetic auxin is used.

[0016] The DNA molecule may be a cDNA clone or a genomic clone isolatedfrom a natural source.

[0017] Methods of isolating and identifying cDNA and genomic clones fromsuch sources are well known in the art. See, e.g. Maniatis et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y., 1982);Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor, N.Y., 1989).

[0018] The phenoxy herbicides are broken down in soil by a variety ofmicroorganisms, including bacteria, yeasts, and fungi from severaltaxonomic groups. See, e.g., Llwellyn and Last, in Herbicide-ResistantCrops, Chapter 10 (Stephen O. Duke, ed.) (CRC Lewis Publishers, NewYork, N.Y., (1996)); Bayley et al., Theor. Appl. Genet., 83, 645-649(1992), Lyon et al., Plant Molec. Biol., 13, 533-540 (1989); Streber etal., J. Bacteriology, 169,2950-2955 (1987); Han and New, Soil Biol.Biochem., 26, 1689-1695 (1994); Donnelly et al., Applied AndEnvironmental Microbiology, 59, 2642-2647 (1993); Loos, in DegradationOf Herbicides, pages 1-49 (Kearney and Kaufman, eds., Marcel Dekker,Inc., New York 1969), and references cited in these references. Specificmicrooganisms include strains of Acinetobacter, Achromobacter,Alcaligenes, Arthrobacter, Corynebacterium, Flavobacterium, Pseudomonas,and Actinomycetes (e.g., Nocardia spp. and Streptomycesviridochromogenes). Additional strains of bacteria, yeast and fungi thatmetabolize phenoxy herbicides can be obtained by methods well known inthe art (e.g., by isolation from soils where the phenoxy herbicides areused or manufactured by the enrichment culture technique (see, e.g.,Loos, in Degradation Of Herbicides, pages 1-49 (Kearney and Kaufman,eds., Marcel Dekker, Inc., New York 1969); Han and New, Soil Biol.Biochem., 26, 1689-1695 (1994)).

[0019] The most well-characterized organisms are strains of Alcaligeneseutrophus, and it is from strains of A. eutrophus that the tfdA geneused to produce 2,4-D-tolerant transgenic plants was isolated.Additional cDNA and genomic clones coding forphenoxy-herbicide-metabolizing enzymes can be obtained frommicroorganisms that metabolize one or more phenoxy herbicides by methodswell known in the art (e.g., in a manner similar to those used toisolate and identify the known tfdA clones). See, e.g., Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1982);Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y. (1989); Streber et al., Bio/Technology, 7, 811-816 (1989);Streber et al., J. Bacteriology, 169, 2950-2955 (1987); Lyon et al.,Plant Molec. Biol., 13, 533-540 (1989); Bayley et al., Theor. Appl.Genet., 83, 645-649 (1992); Perkins and Lurquin, J. Bacteriology, 170,5669-5672 (1988); Llewellyn and Last, in Herbicide-Resistant CropsChapter 10, pages 159-174 (Duke, ed., CRC Lewis Publishers 1996); Lastand Llewellyn, Weed Science, 47, 401-404 (1999); U.S. Pat. Nos.5,608,147, 6,100,446 and 6,153,401; and PCT application WO 95/18862. Inaddition, isolated clones, portions of them, or sequences from themcould be used as probes to identify and isolate additional clones. See,e.g., Perkins and Lurquin, J. Bacteriology, 170, 5669-5672 (1988);Bayley et al., Theor. Appl. Genet., 83, 645-649 (1992); U.S. Pat. Nos.6,100,446 and 6,153,401. See also Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y. (1982), Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1989).

[0020] The DNA molecules comprising DNA encoding an enzyme or enzymeswhich metabolize(s) at least one synthetic auxin can also be fully orpartially chemically synthesized using the sequences of isolated clones.To do so, a cDNA or genomic clone, obtained as described in the previousparagraph, is sequenced by methods well known in the art. See, e.g.,Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y. (1982), Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y. (1989). A synthetic DNA sequencecomprising the coding sequence of the cDNA or genomic clone can be fullyor partially chemically synthesized using methods well known in the art.See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y. (1982), Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y. (1989). For instance, DNAsequences may be synthesized by phosphoamidite chemistry in an automatedDNA synthesizer. Also, the sequence of the tfdA gene from A. eutrophusJMP134 is publically available (see Streber et al., J. Bacteriology,169, 2950-2955 (1987), U.S. Pat. Nos. 6,100,446 and 6,153,401, andGenBank (accession number M 16730)), and a synthetic DNA sequencecomprising the coding sequence of the A. eutrophus tfdA gene can also befully or partially chemically synthesized.

[0021] Chemical synthesis has a number of advantages. For instance,using chemical synthesis, the sequence of the DNA molecule or itsencoded protein can be readily changed to, e.g., optimize expression(e.g., eliminate mRNA secondary structures that interfere withtranscription or translation, eliminate undesired potentialpolyadenylation sequences, and alter the A+T and G+C content), addunique restriction sites at convenient points, delete protease cleavagesites, etc. In particular, chemical synthesis is desirable becausecodons preferred by the plant in which the DNA sequence will beexpressed can be used to optimize expression. Not all of the codons needto be changed to obtain improved expression, but preferably at least thecodons least preferred by the plant are changed to plant-preferredcodons. “Codons least preferred by the plant” are those codons in theheterologous DNA sequence that are used least by the plant in questionto encode a particular amino acid. “Plant-preferred codons” are codonswhich are used more frequently by a plant to encode a particular aminoacid than is the codon encoding that amino acid in the heterologous DNAsequence. Preferably, the plant-preferred codon is the codon used mostfrequently by the plant to encode the amino acid. The plant codon usagemay be that of plants in general, a class of plants (e.g.,dicotyledonous plants), a specific type of plant (e.g., tobacco,soybeans, cotton or tomatoes), etc. The codon usage or preferences of aplant or plants can be deduced by methods known in the art. See, e.g.,Maximizing Gene Expression, pages 225-85 (Reznikoff & Gold, eds., 1986),Perlak, et al., Proc. Natl. Acad. Sci. USA, 88, 3324-3328 (1991), PCT WO97/31115, PCT WO 97/11086, EP 646643, EP 553494, and U.S. Pat. Nos.5,689,052, 5,567,862, 5,567,600, 5,552,299 and 5,017,692. For instance,the codons used by the plant or plants to encode all of the differentamino acids in a selection of proteins expressed by the plant or plants,preferably those proteins which are highly expressed, are tabulated.This can be done manually or using software designed for this purpose(see PCT application WO 97/11086). Preferably, greater than about 50%,most preferably at least about 80%, of the codons of the heterologousDNA sequence are changed to plant-preferred codons.

[0022] In addition, DNA molecules comprising DNA coding for mutantenzymes that metabolize auxin herbicides can be used. Such mutantenzymes would have an amino acid sequence which is the same as that of anaturally-occurring enzyme, such as the dioxygenases produced by the A.eutrophus tfdA clones, except that one or more amino acids is added,deleted, or substituted for the amino acids of the naturally-occurringenzyme. DNA coding for such mutant enzymes can be prepared using, forexample, oligonucleotide-directed mutagenesis, linker-scanningmutagenesis, mutagenesis using the polymerase chain reaction, chemicalsynthesis, and the like. See Ausubel et al. (eds.), Current Protocols InMolecular Biology (Wiley Interscience 1990) and McPherson (ed.),Directed Mutagenesis: A Practical Approach (IRL Press 1991).

[0023] “DNA constructs” are defined herein to be constructed(non-naturally occurring) DNA molecules useful for introducing DNA intohost cells, and the term includes chimeric genes, expression cassettes,and vectors. DNA constructs for use in the present invention compriseDNA coding for an auxin herbicide-metabolizing enzyme or enzymesoperatively linked to expression control sequences.

[0024] As used herein “operatively linked” refers to the linking of DNAsequences (including the order of the sequences, the orientation of thesequences, and the relative spacing of the various sequences) in such amanner that the encoded proteins are expressed. Methods of operativelylinking expression control sequences to coding sequences are well knownin the art. See, e.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y. (1982), Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1989).

[0025] “Expression control sequences” are DNA sequences involved in anyway in the control of transcription or translation. Suitable expressioncontrol sequences and methods of making and using them are well known inthe art.

[0026] The expression control sequences must include a promoter. Thepromoter may be any DNA sequence which shows transcriptional activity inthe chosen plant(s). The promoter may be inducible or constitutive. Itmay be naturally-occurring, may be composed of portions of variousnaturally-occuring promoters, or may be partially or totally synthetic.Guidance for the design of promoters is provided by studies of promoterstructure, such as that of Harley and Reynolds, Nucleic Acids Res., 15,2343-61 (1987). Also, the location of the promoter relative to thetranscription start may be optimized. See, e.g., Roberts, et al., Proc.Natl Acad. Sci. USA, 76, 760-4 (1979). Many suitable promoters are wellknown in the art.

[0027] For instance, suitable constitutive promoters for use in plantsinclude: the promoters from plant viruses, such as the full-lengthtranscript promoter from peanut chlorotic streak virus (U.S. Pat. No.5,850,019), the 35S promoter from cauliflower mosaic virus (Odell etal., Nature 313:810-812 (1985), promoters of Chlorella virusmethyltransferase genes (U.S. Pat. No. 5,563,328), and the full-lengthtranscript promoter from figwort mosaic virus (U.S. Pat. No. 5,378,619);the promoters from such genes as rice actin (McElroy et al., Plant Cell2:163-171 (1990)), ubiquitin (Christensen et al., Plant Mol. Biol.12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689(1992)), pEMU (Last et al., Theor. Appl. Genet. 81:581-588 (1991)), MAS(Velten et al., EMBO J. 3:2723-2730 (1984)), maize H3 histone (Lepetitet al., Mol. Gen. Genet. 231:276-285 (1992) and Atanassova et al., PlantJournal 2(3):291-300 (1992)), Brassica napus ALS3 (PCT application WO97/41228); and promoters of various Agrobacterium genes (see U.S. Pat.Nos. 4,771,002, 5,102,796, 5,182,200, 5,428,147).

[0028] Suitable inducible promoters for use in plants include: thepromoter from the ACE1 system which responds to copper (Mett et al. PNAS90:4567-4571 (1993)); the promoter of the maize In2 gene which respondsto benzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen.Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics243:32-38 (1994)), and the promoter of the Tet repressor from Tn10 (Gatzet al., Mol. Gen. Genet. 227:229-237(1991). A particularly preferredinducible promoterforuse in plants is one that responds to an inducingagent to which plants do not normally respond. An exemplary induciblepromoter of this type is the inducible promoter from a steroid hormonegene, the transcriptional activity of which is induced by aglucocorticosteroid hormone. Schena et al., Proc. Natl. Acad. Sci. USA88:10421 (1991). Other inducible promoters for use in plants aredescribed in EP 332104, PCT WO 93/21334 and PCT WO 97/06269.

[0029] Finally, promoters composed of portions of other promoters andpartially or totally synthetic promoters can be used. See, e.g., Ni etal., Plant J., 7:661-676 (1995) and PCT WO 95/14098 describing suchpromoters for use in plants.

[0030] The promoter may include, or be modified to include, one or moreenhancer elements. Preferably, the promoter will include a plurality ofenhancer elements. Promoters containing enhancer elements provide forhigher levels of transcription as compared to promoters which do notinclude them. Suitable enhancer elements for use in plants include theenhancer element from the full-length transcript promoter of peanutchlorotic streak virus (U.S. Pat. No. 5,850,019), the 35S enhancerelement from cauliflower mosaic virus (U.S. Pat. Nos. 5,106,739 and5,164,316) and the enhancer element from figwort mosaic virus (Maiti etal., Transgenic Res., 6, 143-156 (1997)). Other suitable enhancers foruse in other cells are known. See PCT WO 96/23898 and Enhancers AndEukaryotic Expression (Cold Spring Harbor Press, Cold Spring Harbor,N.Y., 1983).

[0031] For efficient expression, the coding sequences are preferablyalso operatively linked to a 3′ untranslated sequence. The 3′untranslated sequence will include a transcription termination sequenceand a polyadenylation sequence. The 3′ untranslated region can beobtained from the flanking regions of genes from Agrobacterium spp.,plant viruses, plants or other eukaryotic cells. Suitable 3′untranslated sequences for use in plants include those of thecauliflower mosaic virus 35S gene, the phaseolin seed storage proteingene, the pea ribulose biphosphate carboxylase small subunit E9 gene,the soybean 7S storage protein genes, the octopine synthase gene, andthe nopaline synthase gene.

[0032] A 5′ untranslated sequence is also employed. The 5′ untranslatedsequence is the portion of an mRNA which extends from the 5′ CAP site tothe translation initiation codon. This region of the mRNA is necessaryfor translation initiation in eukaryotes and plays a role in theregulation of gene expression. Suitable 5′ untranslated regions for usein plants include those of alfalfa mosaic virus, cucumber mosaic viruscoat protein gene, and tobacco mosaic virus.

[0033] As noted above, the DNA construct may be a vector. The vector maycontain one or more replication systems which allow it to replicate inhost cells. Self-replicating vectors include plasmids, cosmids and viralvectors. Alternatively, the vector may be an integrating vector whichallows the integration into the host cell's chromosome of the sequencecoding for an auxin herbicide-degrading enzyme. The vector desirablyalso has unique restriction sites for the insertion of DNA sequences. Ifa vector does not have unique restriction sites, it may be modified tointroduce or eliminate restriction sites to make it more suitable forfurther manipulations.

[0034] The DNA constructs of the invention can be used to transform anytype of plant cells (see below). A genetic marker must be used forselecting transformed plant cells (“a selection marker”). Selectionmarkers typically allow transformed cells to be recovered by negativeselection (i.e., inhibiting growth of cells that do not contain theselection marker) or by screening for a product encoded by the selectionmarker.

[0035] The most commonly used selectable marker gene for planttransformation is the neomycin phosphotransferase II (nptII) gene,isolated from Tn5, which, when placed under the control of plantexpression control sequences, confers resistance to kanamycin. Fraley etal., Proc. Natl. Acad. Sci. USA, 80:4803 (1983). Another commonly usedselectable marker gene is the hygromycin phosphotransferase gene whichconfers resistance to the antibiotic hygromycin. Vanden Elzen et al.,Plant Mol. Biol., 5:299 (1985).

[0036] Additional selectable marker genes of bacterial origin thatconfer resistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,and the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet. 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol.7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990), Hinchee et al., Bio/Technology, 6:915-922 (1988); Stalker etal., J. Biol. Chem., 263:6310-6314 (1988), and Stalker et al., Science242:419-423 (1988).

[0037] Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

[0038] Commonly used genes for screening presumptively transformed cellsinclude β-glucuronidase (GUS), β-galactosidase, luciferase, andchloramphenicol acetyltransferase. Jefferson, R. A., Plant Mol. Biol.Rep. 5:387 (1987)., Teeri et al., EMBO J. 8:343 (1989), Koncz et al.,Proc. Natl. Acad. Sci. USA 84:131 (1987), De Block et al., EMBO J.3:1681 (1984), green fluorescent protein (GFP) (Chalfie et al., Science263:802 (1994), Haseloff et al., TIG 11:328-329 (1995) and PCTapplication WO 97/41228). Another approach to the identification ofrelatively rare transformation events has been use of a gene thatencodes a dominant constitutive regulator of the Zea mays anthocyaninpigmentation pathway. Ludwig et al., Science 247:449 (1990).

[0039] Selection based on auxin herbicide tolerance or auxin herbicidemetabolism can be used in the production of auxin herbicide-tolerantplants, in which case the use of another selection marker may not benecessary. The preferred auxin herbicides are 2,4-D and its salts(including amine salts) and esters. “Tolerance” in this context meansthat transformed plant cells are able to grow (survive, proliferate andregenerate into plants) when placed in culture medium containing a levelof the auxin herbicide that prevents untransformed cells from doing so.“Tolerance” also means that transformed plants are able to grow afterapplication of an amount of an auxin herbicide that inhibits the growthof untransformed plants.

[0040] Methods of selecting transformed plant cells are well known inthe art. Briefly, at least some of the plant cells in a population ofplant cells (e.g., an explant or an embryonic suspension culture) aretransformed with a DNA construct according to the invention providingfor auxin herbicide metabolism (see below for transformation methods).The resulting population of plant cells is placed in culture mediumcontaining the auxin herbicide at a concentration selected so thattransformed plant cells will grow, whereas untransformed plant cellswill not. Suitable concentrations of auxin herbicide can be determinedempirically as is known in the art. See, e.g., U.S. Pat. No. 5,608,147.See also the Examples below. At least in the case of 2,4-D, this amountmay further need to be an amount which inhibits adventitious shootformation from untransformed plant cells and allows adventitious shootformation from transformed plant cells. See U.S. Pat. No. 5,608,147 andPCT application WO 95/18862. In general, 2,4-D should be present in anamount ranging from about 0.001 mg/l to about 5 mg/l culture medium,preferably from about 0.01 mg/l to 0.2 mg/l culture medium.

[0041] Methods of selecting transformed plants are also known in theart. Briefly, an auxin herbicide is applied to a population of plantswhich may comprise one or more transgenic plants comprising a DNAconstruct according to the invention providing for auxin herbicidemetabolism. The amount of auxin herbicide is selected so thattransformed plants will grow, and the growth of untransformed plantswill be inhibited. The level of inhibition must be sufficient so thattransformed and untransformed plants can be readily distinguished (i.e.,inhibition must be statistically significant). Such amounts can bedetermined empirically as is known in the art. See also Crop ProtectionReference (Chemical & Pharmaceutical Press, Inc., New York, N.Y.,11^(th) ed., 1995) and the Examples below.

[0042] The DNA constructs of the invention can be used to transform avariety of plant cells (see below). The synthetic DNA sequence codingfor the auxin herbicide-metabolizing enzyme and the selection marker, ifa separate selection marker is used, may be on the same or different DNAconstructs. Preferably, they are arranged on a single DNA construct as atranscription unit so that all of the coding sequences are expressedtogether.

[0043] Suitable host cells include plant cells of any kind (see below).Preferably, the plant cell is one that does not normally metabolizeauxin herbicides. However, the present invention can also be used toincrease the level of metabolism of auxin herbicides in plants thatnormally metabolize such herbicides.

[0044] Methods of transforming plants are well known in the art andinclude biological and physical transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp.67-88. In addition, vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick, B. R. andThompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 89-119.

[0045] The most widely utilized method for introducing an expressionvector into plants is based on the natural transformation system ofAgrobacterium. See, for example, Horsch et al., Science 227:1229 (1985).A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteriawhich genetically transform plant cells. The Ti and Ri plasmids of A.tumefaciens and A. rhizogenes, respectively, carry genes responsible forgenetic transformation of the plant. See, for example, Kado, C. I.,Crit. Rev. Plant. Sci. 10:1 (1991). Descriptions of Agrobacterium vectorsystems and methods for Agrobacterium-mediated gene transfer areprovided by numerous references. See, for example, Horsch et al.,Science 227:1229 (1985), Hoekema et al., Nature 303:179 (1983), deFramond et al., Bio/Technology 1:262 (1983), Jordan et al., Plant CellReports 7:281-284 (1988), Leple et al., Plant Cell Reports 11:137-141(1992), Stomp et al., Plant Physiol. 92:1226-1232 (1990), Knauf et al.,Plasmid 8:45-54 (1982)), Gruber et al., supra, Miki et al., supra,Moloney et al., Plant Cell Reports 8:238 (1989), PCT applicationsWO84/02913, WO84/02919 and WO84/02920, EP 116,718, and U.S. Pat. Nos.4,940,838, 5,464,763, and 5,929,300.

[0046] A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles. The expression vector is introduced intoplant tissues with a biolistic device that accelerates themicroprojectiles to speeds sufficient to penetrate plant cell walls andmembranes. Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J.C., Trends Biotech. 6:299 (1988), Sanford, J. C., Physiol. Plant 79:206(1990), Klein et al., Biotechnology 10:268 (1992); Klein et al., Nature,327:70-73 (1987).

[0047] Another method for physical delivery of DNA to plants issonication of target cells. Zhang et al., Bio/Technology 9:996 (1991).Alternatively, liposome or spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes et al., EMBO J.,4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. USA 84:3962(1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-ornithine have also been reported. Hain etal., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant CellPhysiol. 23:451 (1982). Electroporation of protoplasts and whole cellsand tissues has also been described. Donn et al., In Abstracts of VIIthInternational Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p.53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992), Spencer etal., Plant Mol. Biol. 24:51-61 (1994), and Fromm et al., Proc. Natl.Acad. Sci. USA 82:5824 (1985). Other techniques include microinjection(Crossway, Mol. Gen. Genetics, 202:179-185 (1985)), polyethylene glycoltransformation (Krens et al., Nature 296:72-74 (1982)), fusion ofprotoplasts with other entities, either minicells, cells, lysosomes, orother fusible lipid-surfaced bodies (Fraley et al., Proc. Natl. Acad.Sci. USA 79:1859-1863 (1982)), and techniques set forth in U.S. Pat. No.5,231,019.

[0048] After selection, transformed plant cells are regenerated intotransgenic plants. Plant regeneration techniques are well known in theart and include those set forth in the Handbook of Plant Cell Culture,Volumes 1-3, Evans et al., eds. Macmillan Publishing Co., New York, N.Y.(1983, 1984,1984, respectively); Predieri and Malavasi, Plant Cell,Tissue, and Organ Culture 17:133-142 (1989); James, D. J., et al., J.Plant Physiol. 132:148-154 (1988); Fasolo, F., et al., Plant Cell,Tissue, and Organ Culture 16:75-87 (1989); Valobra and James, PlantCell, Tissue, and Organ Culture 21:51-54 (1990); Srivastava, P. S., etal., Plant Science 42:209-214 (1985); Rowland and Ogden, Hort. Science27:1127-1129 (1992); Park and Son, Plant Cell, Tissue, and Organ Culture15:95-105 (1988); Noh and Minocha, Plant Cell Reports 5:464-467 (1986);Brand and Lineberger, Plant Science 57:173-179 (1988); Bozhkov, P. V. etal., Plant Cell Reports 11:386-389 (1992); Kvaalen and von Arnold, PlantCell, Tissue, and Organ Culture 27:49-57 (1991); Tremblay and Tremblay,Plant Cell Tissue, and Organ Culture 27:95-103 (1991); Gupta andPullman, U.S. Pat. No. 5,036,007; Michler and Bauer, Plant Science77:111-118 (1991); Wetzstein, H. Y., et al., Plant Science 64:193-201(1989); McGranahan, G. H., et al., Bio/Technology 6:800-804 (1988);Gingas, V. M., Hort. Science 26:1217-1218 (1991); Chalupa, V., PlantCell Reports 9:398-401 (1990); Gingas and Lineberger, Plant Cell,Tissue, and Organ Culture 17:191-203 (1989); Bureno, M. A., et al.,Phys. Plant. 85:30-34 (1992); and Roberts, D. R., et al., Can. J. Bot.68:1086-1090 (1990), and U.S. Pat. No. 5,608,147.

[0049] Transgenic auxin herbicide-tolerant plants of any type may beproduced. In particular, dicotyledonous crop plants, including beans,soybeans, cotton, peas, potatoes, sunflowers, tomatoes, tobacco, andfruit trees, that are currently known to be injured or killed by auxinherbicides, can be transformed so that they become tolerant to theseherbicides and produce greater yields of their crops. Monocotyledonouscrop plants, such as corn, sorghum, small grains, sugarcane, asparagus,and grasses, which are less sensitive to auxin herbicides thandicotyledonous plants can also be transformed to increase theirtolerance to these herbicides and to increase the yields of their crops.Most crop plants (e.g., soybeans, cotton, tobacco and tomatoes) areannuals, by which it is meant that they typically grow and produce theircrops in a single growing season. Other crop plants (e.g., fruit treesand grasses) are perennials, by which is it meant that the plants growand produce crops for more than a single growing season, generallyseveral years.

[0050] To obtain an increased yield of a crop, transgenic crop plantstolerant to at least one auxin herbicide are grown in the normal mannerto produce the crop. During the growth of the crop, an effective amountof an auxin herbicide to which the transgenic plants are tolerant isapplied to the transgenic plants. The auxin herbicides are applied bymethods well known in the art (see Crop Protection Reference (Chemical &Pharmaceutical Press, Inc., New York, N.Y., 11 th ed., 1995)). Thetiming of the application(s) of the herbicide, the number ofapplications of the herbicide per growing season, and adequate andoptimal amounts of the herbicide to be applied can be determinedempirically, and doing so is within the skill in the art. It has beenfound that application of the auxin herbicide at the seeding/germinationstage should probably be avoided. It has also been found that auxinherbicides can be applied multiple times. It has further been found thatan amount of a phenoxy herbicide up to the amounts normally applied tocontrol dicotyledonous weeds (see Crop Protection Reference (Chemical &Pharmaceutical Press, Inc., New York, N.Y., 11th ed., 1995)) can be usedwithout harm to, and with increased yields of, transgenic tobacco andtomato plants (see the Examples below). Thus, the present method canalso provide for weed control during the growth of crops from whichincreased yields will be obtained. By “increased yield” is meant thattransgenic plants to which an auxin herbicide is applied produce anincreased yield of a crop as compared to non-transgenic plants of thesame type treated in the same manner (i.e., having the same auxinherbicide applied the same number of times and at the same times, etc.).

[0051] In a particularly preferred embodiment of the invention, thetransgenic plants comprise DNA encoding an enzyme that metabolizes2,4-D. Preferably, the DNA encodes the dioxygenase encoded by the tfdAgene which has been isolated from strains of A. eutrophus. It has beenfound that at least the bacterial start codon of the tfdA codingsequence must be replaced by a plant-preferred codon. See, e.g.,Llewellyn and Last, in Herbicide-Resistant Crops Chapter 10, pages159-174 (Duke, ed., CRC Lewis Publishers 1996). Preferably, additionalbacterial codons of the tfdA coding sequence are replaced byplant-preferred codons to obtain better expression of the encodeddioxygenase. Plants expressing the dioxygenase encoded by the tfdI genehave been found to be tolerant to 2,4-D; they have also been found to betolerant to 2,4-DB and MCPA, although at lower levels of herbicide thanfor 2,4-D (data not shown). Plants expressing this enzyme have also beenreported to be tolerant to 4-CPA, but not to be tolerant to 2,4,5-T andphenoxypropionic acid herbicides. See, e.g., Llewellyn and Last, inHerbicide-Resistant Crops Chapter 10, pages 159-174 (Duke, ed., CRCLewis Publishers 1996) and U.S. Pat. Nos. 6,100,446 and 6,153,401. Mostimportant, plants expressing the dioxygenase encoded by the tfdA genehave been found to produce improved crop yields when treated with 2,4-D.

[0052] The following examples are provided for the purpose ofillustration and are not intended to limit the scope of the presentinvention.

EXAMPLES Example 1 Increased Yields of Vegetative Matter by Applicationof 2,4-D to Transgenic Tobacco Plants

[0053] A. Experimental Design

[0054] The use of exogenously supplied synthetic auxins on agronomiccrops to elicit direct non-herbicidal effects, with an emphasis on thepotential enhancement of yield, was assessed usinggenetically-engineered (GE) tobacco plant lines expressing syntheticauxin metabolizing genes. Non-GE controls were used, permittingseparation of the herbicidal and biological properties of theexogenously supplied synthetic auxins. Tobacco was utilized to model drymatter yield.

[0055] The research was conducted in a single phase entailingcharacterization of response variables when plants were subjected tomaximum (1×rate=1 pound/acre of active ingredient) and low (¼×=4ounces/acre of active ingredient) rates of exogenously applied2,4-dichlorophenoxyacetic acid (2,4-D). GE lines of tobacco (dark firedcultivar “KY 160”) expressing a synthetic gene for auxin metabolism(SAD1 or SAD2) were used, along with non-GE lines of the same genotypeas control material. Commercial tobacco production has long utilizeddecapitation (“topping”) to initiate a predictable increase in leafweight, thickness, and dry matter yield. This yield increase isdependent upon variables, such as genotype and fertility. By utilizingcontrols of the same genotype and a fixed level of fertility,significant yield variation responses should be attributable to thetreatment (exogenous auxin herbicide application).

[0056] The overall experimental design was comprised of three parallelcompletely randomized designs with spray rate and genotype as the mainfactors. Three replications were performed for each treatment withineach experiment. Each replication was the average measurement of sixplants. The tobacco lines were allowed to advance to the 6-8 leaf stage.Upon reaching this stage, the primary meristem was removed along withlower leaves so that only 4 leaves remained. Plants were then sprayedwith herbicide and permitted to grow for an additional 21 days. At theend of this period, dry matter yields for both leaf and the whole plantwere measured.

[0057] Yield data recorded for tobacco were leaf dry matter, leafnumber, and total dry matter. Corrected plant yield and corrected leafyield were calculated for each replication as the leaf dry matterdivided by leaf number then multiplied by the expected number of leaves(6 plants×4 leaves=24). This corrected leaf yield was then added to stemyield fraction (arrived at by subtracting actual leaf yield from totalplant yield) to create a corrected total plant yield. All tobacco yieldmeasurements were recorded in grams after a 7-day drying period inambient air dryers.

[0058] B. Transformation of Tobacco to Produce Genetically-EngineeredLines

[0059] 1. Binary Plasmids for Transformation

[0060] The binary plasmids used for transformation were provided by Dr.Mel Oliver (USDA-ARS, Lubbock, Tex.). They were prepared by Dr. Oliverand colleagues as described below. Also see co-pending application No.60/335,463, filed on Oct. 24, 2001, entitled “Synthetic HerbicideResistance Gene”, the complete disclosure of which is incorporatedherein by reference.

[0061] The DNA sequence of a 2,4-D dioxygenase (also often referred toas a monooxygenase) gene isolated from Alcaligenes eutrophus wasobtained from the sequence database GenBank (accession number M16730).From this DNA sequence, the amino acid sequence of the protein coded forby the single open-reading frame (ORF) was determined [SEQ ID NO:1]. Acodon usage table reflecting dicotyledonous ORFs was derived from acomposite selection of random cDNA sequences from cotton, Arabidopsisand tobacco extracted from the GenBank database. Using theplant-specific codon usage table, the derived primary amino acidsequence of the bacterial 2,4-D dioxygenase was converted into DNAcoding sequences that reflected the codon preferences of dicotyledonousplants [SEQ ID NO:2].

[0062] The synthetic plant-optimized 2,4-D dioxygenase ORF [SEQ ID NO:2]was then used to design a 2,4-D dioxygenase gene capable of efficientexpression in transgenic plants. This synthetic gene was designated asSAD (Synthetic gene Adapted for Dicots). In order to generate atranslatable transcript once the gene had been constructed and insertedinto a plant genome, a 5′ untranslated leader sequence representing the5′ untranslated leader sequence from the 35S transcript of alfalfamosaic virus (AMV; Gallie et al., Nucleic Acids Res., 15:8693-8711(1987)) was incorporated into the design of the synthetic gene. Inaddition, a signature sequence, encoding Cys Ala Gly, was added to the3′ end of the synthetic coding region. Finally, for ease of cloning, thedesigned sequence included a HindIII-specific overhang at the 5′ end anda SalI-specific overhang at the 3′ end. The complete designed sequencefor the synthetic portion of the SAD gene is SEQ ID NO:3.

[0063] To construct the designed synthetic portions of the SAD gene,each sequence was dissected into overlapping oligonucleotides, twelveoligonucleotides for each of the two strands resulting in a total oftwenty-four oligonucleotides. The oligonucleotides were synthesizedusing standard phosphoramidite chemistry by Integrated DNA Technologies,Inc., Coralville, Iowa. The synthetic DNA molecules were assembled usinga procedure based upon the protocol described by Sutton et al. 1995published on the World Wide Web (www.epicentre.com) using Ampliligase™thermostable ligase (Epicentre Technologies Inc., Madison, Wis.).Oligonucleotides were first phosphorylated using T4 polynucleotidekinase (Invitrogen Life Technologies, Carlsbad, Calif.) as mixtures ofupper and lower strand oligonucleotides. Each mixture contained 10pmoles of each oligonucleotide, 70 mM Tris/HCl pH 7.6, 10 mM MgCl₂, 5 mMdithiothreitol (DTT), 0.1 mM ATP, and 10 units of T4 polynucleotidekinase, for a total volume of 25 μl. Phosphorylation was achieved byincubation of the mixtures at 37° C. for 30 minutes, followed by adenaturing incubation at 70° C. for 10 minutes. To anneal and ligate theoligonucleotides, each reaction mixture was retreated at 70° C. for 10minutes in a thermocycler and subsequently cooled to 65° C. over a10-minute period. To each mixture, 65 μl of water, 10 μl of10×Ampliligase buffer (Epicentre Technologies), and 2 μl of Ampliligase(5 units/μl) were added sequentially, and the temperature was reduced to40° C. over a three hour period.

[0064] At this stage, in order to improve the efficiency of cloning, thecomplete synthetic DNA sequence for SAD was recovered from itsannealing/ligation reactions by polymerase chain reaction (PCR) in an MJResearch Inc. (Waltham, Mass.) Model PTC-100 Thermocycler using AmplitaqGold™ DNA polymerase under conditions supplied by the manufacturer,Perkin Elmer Life Sciences (Boston, Mass.). The PCR primers used for therecovery of each sequence were a 28mer representing the 5′ end of theAMV leader sequence and a 25mer specific for the 3′ end of the SADsequence. PCR fragments corresponding to the appropriate size of 918 bpwere gel purified as described in Ausubel et al., Current Protocols InMolecular Biology (Green/Wiley Interscience, New York, 1989) and clonedbetween two XcmI restriction sites in pUCR19, a modified pUC19 vectordesigned for rapid cloning of PCR fragments using T overhangs generatedby XcmI digestion (described in O'Mahony and Oliver, Plant MolecularBiology, 39:809-821 (1999)) to generate the plasmid pUCRsynSAD. Oncecloned into this vector, the insert was sequenced to verify the sequenceintegrity of the designed synthetic portion of the SAD gene. DNAsequencing was performed by use of a dRhodamine Terminator CycleSequencing kit (PE Applied Biosystems, Foster City, Calif.) according tothe manufacturer's instructions. Sequence reactions were analyzed usinga Perkin Elmer/ABI Prism 310 automated sequencer.

[0065] For the generation of a complete and plant-competent SAD gene,the synthetic portions of the SAD gene contained in pUCRsynSAD wereremoved by first releasing the 5′ end of the synthetic sequence bydigestion with XbaI and filling in the overhang with DNA polymerase I(Klenow large fragment) followed by digestion with KpnI. This fragmentwas ligated into the plasmid pProPClSV, a pUC19 plasmid containing anenhanced Peanut Chlorotic Streak Virus (PClSV) promoter derived frompKLP36 (described by Maiti and Shepherd, Biochem. Biophys. Res. Com.,244:440-444 (1998)) by cutting first with NcoI, treating with DNApolymerase I (Klenow large fragment) to fill in the generated overhang,and subsequently cutting with KpnI. This generated the plasmidpProPClSV-SAD within which the synthetic portion of the SAD gene,including the 5′ AMV leader and 3′ region coding for the Cys Ala Glysignature, is directly linked to the 3′ end of the PClSV promoter (FIG.1). This plasmid served as the source for the PClSV-SAD construction forinsertion into the binary vectors for final gene construction prior tointroduction into Agrobacterium for plant transformation.

[0066] Two binary vectors were chosen for final SAD gene construction,pPZP211-PNPT-311 g7 (FIG. 2) and pPZP211-PNPT-512g7 (FIG. 3). These twovectors are based on the pPZP family of vectors described byHajdukiewicz et al., Plant Molec. Biol., 25:989-994 (1994) and arepPZP211 derivatives in which the neomycin phosphotransferase II (NPTII)gene for kanamycin resistance is driven by the PClSV promoter and a g7polyA termination sequence is placed adjacent to a multicloning site(MCS, FIGS. 2 and 3). The only difference between these two vectors isthe position of the MCS relative to the g7 polyA termination sequence.The g7 polyA termination sequence is the 3′ polyA termination signalfrom gene 7 within the octopine T-Left region of an octopineAgrobacterium tumefaciens Ti plasmid and was isolated as an EcoRI-SalIfragment from pAP2034 (Velten and Schell, Nucleic Acids, 13:6981-6998(1985)).

[0067] The complete SAD gene was constructed by removal of the PClSV-SADsequence from pProPClSV-SAD as a HindIII-SmaI fragment and insertioninto both pPZP211-PNPT-311 g7 and pPZP211-PNPT-512g7 that were first cutwith BamHI, treated with DNA polymerase I (Klenow large fragment) tofill in the overhanging sequence, and subsequently digested withHindIII. These reactions generated the two vectors, pPZP211-PNPT-311-SAD(FIG. 4) and pPZP211-PNPT-512-SAD (FIG. 5), that contained the fullplant expressible SAD gene in one of two orientations with respect tothe PClSV-NPTII-35SpolyA construct. This design for insertion of the SADgene into plant genomes was implemented because of uncertainty as to theeffect of having two PClSV promoter sequences in the same plasmid onboth transformation and effective transmission of the expressed trait.By putting the SAD gene in the vectors such that the PClSV promoterswere inserted as both direct and inverted repeats, the possibility of anegative outcome could be avoided.

[0068] After construction, the SAD genes in each vector were sequencedas described above to ensure fidelity. This sequencing revealed that, inthe construction of pProPClSV-SAD, an out-of-frame ATG codon wasintroduced into the 5′ untranslated leader sequence. The presence ofthis ATG codon could alter the translatability of the transcript thatwould be synthesized from the SAD gene and so was deleted by PCRmutagenesis to restore the normal AMV leader sequence. Following repair,the sequence was rechecked for fidelity. The original SAD genecontaining the out-of-frame ATG was labelled SAD1 (since sometransformation experiments had begun using this construct). The repairedSAD gene is referred to as SAD2.

[0069] 2. Transformation of Tobacco

[0070] Plasmids pPZP211-PNPT-311-SAD1 and pPZP211-PNPT-311-SAD2 wereeach transformed into Agrobacterium tumefaciens strain EHA 105 byelectroporation (Dulk-Ras and Hooykaas, in Plant Cell Electroporationand Electrofusion Protocols (Nickloff, ed., Humana Press, Inc., Totowa,N.J.). Similar results have been obtained with plasmidspPZP211-PNPT-512-SAD1 and pPZP211-PNPT-512-SAD2 (data not shown), andthey can be used in place of pPZP211-PNPT-311-SAD1 andpPZP211-PNPT-311-SAD2.

[0071]Nicotiana tabacum cultivar KY 160 (University of KentuckyExperiment Station) was used for all experiments. Seeds were surfacesterilized and germinated on minimal medium [MS salts (Murashige andSkoog, Physiol. Plant, 15:473-497 (1962)); B5 vitamins (Gamborg et al.,Experimental Cell Research, 50:151-158 (1968)), 3% sucrose, pH 5.8,solidified with 0.8% agar]. Leaves from 3-4 week old seedlings wereexcised, wounded with a number II scalpel blade, and exposed to A.tumefacicens carrying either pPZP211-PNPT-311-SAD1 orpPZP211-PNPT-311-SAD2. After exposure to A. tumefaciens, explants wereco-cultured on shooting medium [MS salts, B5 vitamins, 3% sucrose, 2.5μg/l 6-benzylaminopurine, 1.0 μg/l indole-3-acetic acid, pH 5.8,solidified with 0.8% agar] overnight at 27° C. Following co-cultivation,explants were transferred to shooting medium amended with 300 mg/lkanamycinmonosulfate and 500 mg/l cefatoxim. Putative transgenic shootsbegan to appear after 21 days, and at 30-50 days, shoots were excisedand placed on minimal medium containing kanamycin monosulfate (300 mg/l)and cefatoxim (500 mg/l) for root development. Once roots wereestablished, the plantlets were transferred into soil in the greenhouseand allowed to flower. Plants were bagged to ensure self-pollination.

[0072] Seed from the T0 plants (T1 seed) was germinated on minimalmedium containing 300 mg/l kanamycin monosulfate to select forkanamycin-resistant (Kan^(R)) plants. Kan^(R) seedlings were transferredto soil for generational advance. Again, plants were bagged to ensureself-pollination. Seed from T1 plants (T2 seed) was germinated as above,and homozygous transgenic lines (lines in which all of the T2 seedlingswere Kan^(R)) were selected for spray assays and yield evaluation.

[0073] C. Spray Assays

[0074] Sterile seeds of putative transgenic tobacco lines weregerminated on minimal medium containing 300 mg/L kanamycin monosulfate.At 21 days post-germination, tobacco seedlings were identified as beingtransgenic if they both remained green and formed secondary roots.Segregating non-transgenic progeny were identified by their chlorosis,bleaching and failure to form strong root systems. Transgenic progeny(green seedlings) were transferred to 4″ azalea pots filled with a 2:2:1mix of ProMix BX (Premier Horticulture Inc., Red Hill, Pa. 18076),Masonry sand, and field soil and allowed to grow for 21 days ongreenhouse benches under a 16:8 light:dark cycle. Then, the tobaccoseedlings were sprayed as described below, except using a 2×rate(equivalent of 32 ounces of 2,4-D, isooctyl ester formulation, per acre)only. Ten days after spraying, the seedlings were evaluated fortolerance, Tolerance was scored as: 0=dead; 1=severe deformationsymptoms; 2=mild to moderate symptoms; and 3=no symptoms.

[0075] D. Yield Assays

[0076] 1. Growth of Plants

[0077] Seed of non-transgenic tobacco lines were germinated on MS03 (MSsalts, B5 vitamins, 3% sucrose) medium. Transgenic seeds were germinatedon the same medium augmented with 300 mg/L kanamycin monosulfate toallow detection of any seedlings failing to carry the transgene. Thirtydays after germination, seedlings were transplanted to the greenhouseinto flats holding 8 trays of 6 plants each for a total of 48 plants.Plants were permitted to grow for approximately 45 days, at which time6-8 leaves had developed. Prior to spraying, the top of the plantretaining the uppermost two leaves was removed (decapitated) resultingin the retention of 4 fully-extended leaves for each plant.

[0078] 2. Spraying of Plants

[0079] For spray assays and for the yield studies, spraying wasperformed in an Allen Machine Works Spray Cabinet. Spray rates wereestablished as the equivalent of 16 ounces of active ingredient per acre(1×rate), 4 ounces active ingredient/acre (¼×rate), and the control (noactive ingredient per acre or OX). The herbicide used was 2,4-D,isooctyl ester formulation (3.9 pounds of active ingredient/gallon) atequivalent rates noted above. To achieve this equivalent 1×field rate,500 microliters of herbicide were diluted into a total volume of 50 ml.The ¼×field rate was achieved by diluting 125 microliters of herbicidein a 50 ml volume. This 50 ml was dispensed at a rate of 358.5 ml perminute in a 15″ band applied by a 8001 EVS nozzle at 29 pounds persquare inch (PSI). This is equivalent to 25 gallons per acre beingapplied at a ground speed of 1.5 MPH. Each replication was sprayedindependently and permitted to dry before being moved back to thegreenhouse for further growth and evaluation.

[0080] 3. Determination of Dry Matter Yields

[0081] Twenty-one days after spraying, tobacco plants were harvested bycutting off all above-ground growth. The leaves were stripped off, andeach plant and its leaves were bagged and dried at 75° C. with forcedair for 36 hours. The plants were removed from bags, the leaves wereweighed, the stems were weighed, and total plant biomass was determinedas the sum of the leaf and stem weights.

[0082] 4. Results

[0083] The results are presented in the following tables. Table 1demonstrates the yield effects attributable to genotype and spray rate.This is possible because the analyses of variance (Table 2) forvegetative yield components in tobacco clearly demonstrated a genotypeby spray rate interaction for all yield response variables measured,except for uncorrected plant yield. This interaction was expected, sincesusceptible non-transgenic control material should show a significantdecrease in yield when treated with a herbicide that the controlmaterial has no tolerance to. When evaluating Table 1, note the positiveyield increase in the transgenic materials. Whenever transgenic materialwas sprayed with either the ¼× or 1×rate of 2,4-D, a significantincrease in yield was produced. This yield increase is likely a result(as noted by the leaf yield, corrected leaf yield, and percentage leafyield responses) of increased leaf tissue and decreased stalk fraction,a highly desirable increase in any crop harvested for foliage.Additionally, non-transgenic control material is also showing this sameincreasing pattern across all values at the lower (¼×) application rate.This clearly shows that, at the lower evaluated ¼×rate, exogenouslyapplied phenoxy herbicide does alter vegetative yield in a positivemanner.

[0084] To summarize, vegetative data clearly demonstrated that theexogenous application of a phenoxy herbicide (in this case 2,4-Disooctyl ester) increased vegetative yield. The data further suggestedthat this yield increase occurred predominantly in the leaf yieldcomponent of total plant yield. This reveals a new and novel use of thisherbicide, in combination with genes conditioning tolerance to it, forproviding demonstrable yield increases. The commercialization of tobaccoor other leaf crops expressing genes for tolerance to phenoxy herbicideswould permit growers to obtain yield increases in crops normallysusceptible to these same herbicides. TABLE 1 Effect of Spray Rates of2,4-D on Dark Fire Tobacco Genotype KY160 Means (in grams) asConditioned by Either Herbicide Metabolic Gene 2,4-D Plant Corrected⁵Leaf Corrected⁵ Percent Genotype Rate⁴ Yield⁵ Plant Yield Yield⁵ LeafYield Leaf Yield⁵ KY 160¹ 0 29.9 30.6 10.5 11.2 36.6% 1/4X 33.1 34.311.0 12.2 35.2% 1X 27.7 28.2 9.0 9.4 33.2% SAD1² 0 38.7 39.3 14.1 14.737.3% 1/4X 43.5 43.9 17.4 17.8 40.5% 1x 42.5 42.7 18.8 19.1 44.7% SAD2³0 40.6 42.4 14.2 16.0 37.5% 1/4X 43.9 45.2 17.8 19.1 42.2% 1X 48.8 51.019.4 21.6 42.2%

[0085] TABLE 2 Statistical Analysis of Yield Effects as Conditioned bySpray Rates on Transgenic and Non-transgenic Dark Fired Tobacco GenotypeKY 160. A) Analysis of Variance for Variable: Plant Yield Source DF Sumof Squares Mean Square F Value Pr > F Genotype 2 1793.299 896.65 42.90.001* Spray Rate 2 155.033 77.52 3.71 0.033* Geno × Spray 4 200.11350.03 2.39 0.066 Error 42 877.877 20.90 R-Square C.V. Root MSE PlantYield Mean 0.709 11.646 4.571 39.255 B) Analysis of Variance forVariable: Plant Corrected Yield Source DF Sum of Squares Mean Square FValue Pr > F Genotype 2 1960.360 980.18 52.68 0.001* Spray Rate 2149.550 74.78 4.02 0.025* Geno × Spray 4 245.748 61.44 3.30 0.019* Error42 781.391 18.60 R-Square C.V. Root MSE Plant Corrected Yield Mean 0.75110.718 4.313 40.244 C) Analysis of Variance for Variable: Leaf YieldSource DF Sum of Squares Mean Square F Value Pr > F Genotype 2 494.749247.37 38.47 0.001* Spray Rate 2 92.513 46.26 7.19 0.021* Geno × Spray 474.535 18.63 2.90 0.033* Error 42 270.042 6.43 R-Square C.V. Root MSELeaf Yield Mean 0.710 16.952 2.535 14.958 D) Analysis of Variance forVariable: Corrected Leaf Yield Source DF Sum of Squares Mean Square FValue Pr > F Genotype 2 564.432 282.22 50.93 0.001* Spray Rate 2 88.52944.27 7.99 0.001* Geno × Spray 4 87.609 21.90 3.95 0.008* Error 42232.747 5.54 R-Square C.V. Root MSE Plant Yield Mean 0.761 14.762 2.35415.946 E) Analysis of Variance for Variable: Percentage Leaf YieldSource DF Sum of Squares Mean Square F Value Pr > F Genotype 2 0.3470.0174 16.05 0.001* Spray Rate 2 0.009 0.0048 4.40 0.018* Geno × Spray 40.018 0.0046 4.24 0.006* Error 42 0.045 0.0011 R-Square C.V. Root MSEPlant Yield Mean 0.579 8.423 0.033 0.390

Example 2 Increased Yield of Fruit as a Result of Application of 2,4-Dto Transgenic Tomato Plants

[0086] A. Experimental Design

[0087] The use of exogenously supplied synthetic auxins on agronomiccrops to elicit direct non-herbicidal effects was further assessed usinggenetically-engineered tomato plant lines expressing a synthetic auxinmetabolizing gene, and non-genetically-engineered controls. Tomato wasutilized to model fruit retention (fruit set) and fruit yield. Thetransgenic tomato line was utilized in the T2 generation with thetransgene fixed in a homozygous fashion. The tomato genotype UC82L wasutilized as the control, as well as the background for tranformationwith the pPZP211-PNPT-311-SAD2 binary plasmid. This genotype was chosenbecause it is more likely to closely approximate the behavior offield-grown tomatoes than some short-life-cycle laboratory strains oftomato.

[0088] In the same manner as for tobacco, the research was conducted ina single phase entailing characterization of response variables whenplants were subjected to the maximum (1×ate=1 pound/acre of activeingredient) and low (¼×=4 ounces/acre of active ingredient) rates ofexogenously applied 2,4-D. The overall experimental design was comprisedof three parallel completely randomized designs with spray rate andgenotype as the main factors. Three replications were performed for eachtreatment within each experiment. Each replication was the average ofthree plants that were harvested over a period of 60 days. Fruit number,fruit weight and flower number were measured directly, with averagefruit weight and average fruit per plant being derived by dividing fruitweight and fruit number within a replication by the number of plants(3). Tomato fruit yield was measured on fresh fruit and recorded ingrams.

[0089] B. Transformation of Tomato to Produce Genetically-EngineeredLines

[0090] The binary plasmid pPZP211-PNPT-311-SAD2 used for transformationwas the same as that used in Example 1, and tomato (Lycopersiconesculentum) cultivar UC82B (a cultivar nearly identical to UC82L) wastransformed by Agrobacterium-mediated transformation of leaf tissue withthe binary plasmid as described in Example 1. Transgenic plants wereproduced from the transformed leaf tissue as described in Example 1.

[0091] C. Yield Assays

[0092] Non-transgenic and transgenic seed were germinated and grown asdescribed in Example 1 for tobacco. The transgenic tomato line wasutilized in the T2 generation with the transgene fixed in a homozygousfashion.

[0093] The plants with 4 fully-extended leaves retained were sprayed asdescribed in Example 1. Each replication (3 plants) was sprayedindependently and permitted to dry before being moved back to thegreenhouse for further growth for 60 days.

[0094] Fruit number, fruit weight, and flower number were measureddirectly during this 60-day period. Tomato fruit yield was measured onfresh fruit and recorded in grams.

[0095] D. Results

[0096] The results are presented in the following tables. The analysesof variance for tomato fruit response variables (Table 3) shows thatsignificant differences were found in total fruit yield, total flowernumber, and average fruit weight between treatments. Genotype wasdetermined to be the significant factor for total fruit yield, which,just as for the tobacco model above, was expected, since non-transgenicmaterial that is susceptible to a herbicide should display reduced vigorwhen treated with that herbicide. What was not expected, but is evidentin Table 4, was the increase in total yield of the transgenic linesexpressing the herbicide tolerance gene. Total flower number was alsodetermined to be significantly different between treatments, butevaluation of fruit per plant (a later measure influenced by flowernumber) did not show any significant differences between treatments.This left average fruit weight as the final remaining significantmeasure affected by the treatments. In the case of average fruit weight,genotype by spray rate interactions were significant, as can be seenfrom Table 3. When the means of the measured traits are compared (Table4), it is clear that the ¼×rate of 2,4-D application resulted in anincrease in average fruit weight for both control and transgenic lines.This increase is most striking in the non-transgenic material, althoughevident also in transgenic material. The ¼×rate also affected totalflower number, reducing the number of flowers in the control materialand increasing the number of flowers in the transgenic lines.

[0097] In summary, the fruit yield data demonstrated that there was apositive benefit in transgenic lines treated with a phenoxy herbicide.This again reveals a new and novel use of these herbicides incombination with genes conditioning tolerance to them for providingdemonstrable yield increases. The commercialization of tomato or otherfruit crops expressing genes for tolerance to phenoxy herbicides wouldpermit growers to obtain yield increases in crops normally susceptibleto these same herbicides.

[0098] In combination, the results of Examples 1 and 2 show that 2,4-Dmetabolizing genes provide tolerance to 2,4-D in both tobacco and tomatoand, in combination with application of phenoxy herbicide, give yieldincreases. The efficacy of these genes in two distinctly different cropsalso demonstrates the potential for use in other crops to increaseforage yield (such as alfalfa) or fruit yield (such as melons). Not onlywill transgenic crops expressing these genes benefit from potentialincreases in yield, these transgenic crops will be applied to capitalizeon the benefits of weed control provided by phenoxy herbicides. TABLE 3Statistical Analysis of Tomato Yield Components As Influenced byGenotype and Spray Rate A) Analysis of Variance for Variable: TomatoTotal Fruit Yield Source DF Sum of Squares Mean Square F Value Pr > FGenotype 1 14402.401 14402.401 6.91 0.025* Spray Rate 2 312.938 156.4690.08 0.928 Geno × Spray 2 4404.824 2202.412 1.06 0.383 Error 1020852.384 2085.238 R-Square C.V. Root MSE Total Fruit Yield Mean 0.47851.423 45.66 88.800 B) Analysis of Variance for Variable: Total TomatoFruit Number Source DF Sum of Squares Mean Square F Value Pr > FGenotype 1 49.000 49.000 3.47 0.092 Spray Rate 2 34.241 17.121 1.210.337 Geno × Spray 2 65.592 32.796 2.32 0.148 Error 10 141.167 14.117R-Square C.V. Root MSE Total Fruit Mean 0.513 53.67 3.76 7.000 C)Analysis of Variance for Variable: Total Flower Number Source DF Sum ofSquares Mean Square F Value Pr > F Genotype 1 100.000 100.000 1.69 0.222Spray Rate 2 376.119 188.059 3.19 0.085 Geno × Spray 2 471.637 235.8154.00 0.053* Error 10 590.000 50.00 R-Square C.V. Root MSE Total FlowerMean 0.616 49.95 7.68 15.375 D) Analysis of Variance for Variable:Average Fruit Weight Source DF Sum of Squares Mean Square F Value Pr > FGenotype 1 4.378 4.378 0.35 0.566 Spray Rate 2 223.890 111.945 9.020.058 Geno × Spray 2 172.352 86.176 6.940 0.013* Error 10 124.107 12.410R-Square C.V. Root MSE Plant Yield Mean 0.763 24.67 3.52 14.281 E)Analysis of Variance for Variable: Averaae Fruit Per Plant Source DF Sumof Squares Mean Square F Value Pr > F Genotype 1 3.063 3.063 3.47 0.092Spray Rate 2 2.140 1.070 1.21 0.337 Geno × Spray 2 4.099 2.049 2.320.148 Error 10 8.822 0.822 R-Square C.V. Root MSE Average Fruit/PlantMean 0.513 53.67 0.94 1.750

[0099] TABLE 4 Genotype by Spray Rate Interaction Means for ResponseVariables Spray Total Total⁵ Fruit per⁶ Total⁷ Fruit⁸ Genotype Rate³Yield⁴ Fruit No. Plant Flowers Weight UC82L¹ 0 77.4 9.7 2.4 26.0 8.0UC82L 1/4X 66.1 3.0 0.8 6.0 26.0 UC82L 1x 35.3 2.3 0.6 4.3 14.1 SAD2² 092.9 7.5 1.9 16.0 12.9 SAD2 1/4X 122.9 8.7 2.2 17.3 14.4 SAD2 1X 131.99.7 2.4 19.7 13.7

Example 3 Increased Yields by Application of 2,4-D to Transgenic CottonPlants

[0100] Transgenic cotton plants (transformed as described in Bayley etal., Theor. Appl. Genet., 83:645-649 (1992)) were grown from transgenicseed obtained from USDA, Lubbock, Tex. The cotton plants were sprayedwith 2,4-D amine at 1 lb of active ingredient per acre. It was foundthat the 2,4-D amine application significantly increased the yield ofcotton. The results are presented in Table 5 below. TABLE 5 COTTONHERBICIDE SEED COTTON PERCENT LINE¹ RATE YIELD² INCREASE X-2600 0 1986 —X-2600 1 lb/acre 2145 8.0 X-2603 0 2597 — X-2603 1 lb/acre 2790 7.4X-2606 0 2566 — X-2606 1 lb/acre 3067 19.5 X-2609 0 2327 — X-2609 1lb/acre 2301 −1.1 X-2614 0 1991 — X-2614 1 lb/acre 2660 33.6 X-2615 02065 — X-2615 1 lb/acre 2380 15.3 X-2619 0 2056 — X-2619 1 lb/acre 22358.7 AVERAGE 13.0

[0101] While various embodiments of the present invention have beendescribed in detail, it is apparent that modifications and adaptationsof those embodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1 3 1 287 PRT Alcaligenes eutrophus 1 Met Ser Val Val Ala Asn Pro LeuHis Pro Leu Phe Ala Ala Gly Val 1 5 10 15 Glu Asp Ile Asp Leu Arg GluAla Leu Gly Ser Thr Glu Val Arg Glu 20 25 30 Ile Glu Arg Leu Met Asp GluLys Ser Val Leu Val Phe Arg Gly Gln 35 40 45 Pro Leu Ser Gln Asp Gln GlnIle Ala Phe Ala Arg Asn Phe Gly Pro 50 55 60 Leu Glu Gly Gly Phe Ile LysVal Asn Gln Arg Pro Ser Arg Phe Lys 65 70 75 80 Tyr Ala Glu Leu Ala AspIle Ser Asn Val Ser Leu Asp Gly Lys Val 85 90 95 Ala Gln Arg Asp Ala ArgGlu Val Val Gly Asn Phe Ala Asn Gln Leu 100 105 110 Trp His Ser Asp SerSer Phe Gln Gln Pro Ala Ala Arg Tyr Ser Met 115 120 125 Leu Ser Ala ValVal Val Pro Pro Ser Gly Gly Asp Thr Glu Phe Cys 130 135 140 Asp Met ArgAla Ala Tyr Asp Ala Leu Pro Arg Asp Leu Gln Ser Glu 145 150 155 160 LeuGlu Gly Leu Arg Ala Glu His Tyr Ala Leu Asn Ser Arg Phe Leu 165 170 175Leu Gly Asp Thr Asp Tyr Ser Glu Ala Gln Arg Asn Ala Met Pro Pro 180 185190 Val Asn Trp Pro Leu Val Arg Thr His Ala Gly Ser Gly Arg Lys Phe 195200 205 Leu Phe Ile Gly Ala His Ala Ser His Val Glu Gly Leu Pro Val Ala210 215 220 Glu Gly Arg Met Leu Leu Ala Glu Leu Leu Glu His Ala Thr GlnArg 225 230 235 240 Glu Phe Val Tyr Arg His Arg Trp Asn Val Gly Asp LeuVal Met Trp 245 250 255 Asp Asn Arg Cys Val Leu His Arg Gly Arg Arg TyrAsp Ile Ser Ala 260 265 270 Arg Arg Glu Leu Arg Arg Ala Thr Thr Leu AspAsp Ala Val Val 275 280 285 2 864 DNA Artificial sequence Dicot ORF fordegradation of 2,4-D 2 atg tct gtt gtt gct aac cct ttg cat cct ttg ttcgct gct gga gtt 48 Met Ser Val Val Ala Asn Pro Leu His Pro Leu Phe AlaAla Gly Val 1 5 10 15 gag gat att gat ctc aga gaa gca ttg ggt tct actgag gtg aga gaa 96 Glu Asp Ile Asp Leu Arg Glu Ala Leu Gly Ser Thr GluVal Arg Glu 20 25 30 att gag aga ctc atg gac gaa aag tca gtt ctc gtt ttcaga ggt caa 144 Ile Glu Arg Leu Met Asp Glu Lys Ser Val Leu Val Phe ArgGly Gln 35 40 45 cca ctc tca cag gat caa cag att gct ttt gct agg aat tttgga cct 192 Pro Leu Ser Gln Asp Gln Gln Ile Ala Phe Ala Arg Asn Phe GlyPro 50 55 60 ttg gag ggt gga ttc atc aaa gtg aac cag aga cca tct agg ttcaaa 240 Leu Glu Gly Gly Phe Ile Lys Val Asn Gln Arg Pro Ser Arg Phe Lys65 70 75 80 tat gct gaa ctc gct gat atc tct aat gtt tca ttg gat ggt aaggtg 288 Tyr Ala Glu Leu Ala Asp Ile Ser Asn Val Ser Leu Asp Gly Lys Val85 90 95 gca caa aga gac gct aga gaa gtt gtg gga aat ttt gca aat caa ttg336 Ala Gln Arg Asp Ala Arg Glu Val Val Gly Asn Phe Ala Asn Gln Leu 100105 110 tgg cat tct gat tct tca ttc caa cag cca gca gct aga tat tct atg384 Trp His Ser Asp Ser Ser Phe Gln Gln Pro Ala Ala Arg Tyr Ser Met 115120 125 ttg tca gct gtt gtt gtg cct cct tct gga ggt gat aca gaa ttt tgt432 Leu Ser Ala Val Val Val Pro Pro Ser Gly Gly Asp Thr Glu Phe Cys 130135 140 gat atg agg gca gct tac gat gct ctc cca agg gat ttg cag tct gaa480 Asp Met Arg Ala Ala Tyr Asp Ala Leu Pro Arg Asp Leu Gln Ser Glu 145150 155 160 ctc gag gga ttg aga gct gaa cat tac gct ttg aac tca aga tttctc 528 Leu Glu Gly Leu Arg Ala Glu His Tyr Ala Leu Asn Ser Arg Phe Leu165 170 175 ttg gga gat act gat tac tca gag gca cag aga aac gct atg cctcct 576 Leu Gly Asp Thr Asp Tyr Ser Glu Ala Gln Arg Asn Ala Met Pro Pro180 185 190 gtt aac tgg cct ctc gtt agg act cat gct ggt tct ggt aga aagttc 624 Val Asn Trp Pro Leu Val Arg Thr His Ala Gly Ser Gly Arg Lys Phe195 200 205 ttg ttt att gga gca cat gct tca cat gtt gag ggt ctc cct gttgct 672 Leu Phe Ile Gly Ala His Ala Ser His Val Glu Gly Leu Pro Val Ala210 215 220 gag gga aga atg ttg ctc gct gaa ttg ctc gaa cat gct act caaaga 720 Glu Gly Arg Met Leu Leu Ala Glu Leu Leu Glu His Ala Thr Gln Arg225 230 235 240 gag ttt gtt tat aga cac aga tgg aat gtt ggt gac ttg gttatg tgg 768 Glu Phe Val Tyr Arg His Arg Trp Asn Val Gly Asp Leu Val MetTrp 245 250 255 gat aat aga tgt gtg ttg cat aga ggt agg aga tat gat atttct gct 816 Asp Asn Arg Cys Val Leu His Arg Gly Arg Arg Tyr Asp Ile SerAla 260 265 270 aga agg gaa ctc aga agg gct act act ttg gat gac gct gttgtt tag 864 Arg Arg Glu Leu Arg Arg Ala Thr Thr Leu Asp Asp Ala Val Val275 280 285 3 918 DNA Artificial sequence Dicot gene for degradation of2,4-D 3 agatcctttt tatttttaat tttctttcaa atacttccag atcc atg tct gtt gtt56 Met Ser Val Val 1 gct aac cct ttg cat cct ttg ttc gct gct gga gtt gaggat att gat 104 Ala Asn Pro Leu His Pro Leu Phe Ala Ala Gly Val Glu AspIle Asp 5 10 15 20 ctc aga gaa gca ttg ggt tct act gag gtg aga gaa attgag aga ctc 152 Leu Arg Glu Ala Leu Gly Ser Thr Glu Val Arg Glu Ile GluArg Leu 25 30 35 atg gac gaa aag tca gtt ctc gtt ttc aga ggt caa cca ctctca cag 200 Met Asp Glu Lys Ser Val Leu Val Phe Arg Gly Gln Pro Leu SerGln 40 45 50 gat caa cag att gct ttt gct agg aat ttt gga cct ttg gag ggtgga 248 Asp Gln Gln Ile Ala Phe Ala Arg Asn Phe Gly Pro Leu Glu Gly Gly55 60 65 ttc atc aaa gtg aac cag aga cca tct agg ttc aaa tat gct gaa ctc296 Phe Ile Lys Val Asn Gln Arg Pro Ser Arg Phe Lys Tyr Ala Glu Leu 7075 80 gct gat atc tct aat gtt tca ttg gat ggt aag gtg gca caa aga gac344 Ala Asp Ile Ser Asn Val Ser Leu Asp Gly Lys Val Ala Gln Arg Asp 8590 95 100 gct aga gaa gtt gtg gga aat ttt gca aat caa ttg tgg cat tctgat 392 Ala Arg Glu Val Val Gly Asn Phe Ala Asn Gln Leu Trp His Ser Asp105 110 115 tct tca ttc caa cag cca gca gct aga tat tct atg ttg tca gctgtt 440 Ser Ser Phe Gln Gln Pro Ala Ala Arg Tyr Ser Met Leu Ser Ala Val120 125 130 gtt gtg cct cct tct gga ggt gat aca gaa ttt tgt gat atg agggca 488 Val Val Pro Pro Ser Gly Gly Asp Thr Glu Phe Cys Asp Met Arg Ala135 140 145 gct tac gat gct ctc cca agg gat ttg cag tct gaa ctc gag ggattg 536 Ala Tyr Asp Ala Leu Pro Arg Asp Leu Gln Ser Glu Leu Glu Gly Leu150 155 160 aga gct gaa cat tac gct ttg aac tca aga ttt ctc ttg gga gatact 584 Arg Ala Glu His Tyr Ala Leu Asn Ser Arg Phe Leu Leu Gly Asp Thr165 170 175 180 gat tac tca gag gca cag aga aac gct atg cct cct gtt aactgg cct 632 Asp Tyr Ser Glu Ala Gln Arg Asn Ala Met Pro Pro Val Asn TrpPro 185 190 195 ctc gtt agg act cat gct ggt tct ggt aga aag ttc ttg tttatt gga 680 Leu Val Arg Thr His Ala Gly Ser Gly Arg Lys Phe Leu Phe IleGly 200 205 210 gca cat gct tca cat gtt gag ggt ctc cct gtt gct gag ggaaga atg 728 Ala His Ala Ser His Val Glu Gly Leu Pro Val Ala Glu Gly ArgMet 215 220 225 ttg ctc gct gaa ttg ctc gaa cat gct act caa aga gag tttgtt tat 776 Leu Leu Ala Glu Leu Leu Glu His Ala Thr Gln Arg Glu Phe ValTyr 230 235 240 aga cac aga tgg aat gtt ggt gac ttg gtt atg tgg gat aataga tgt 824 Arg His Arg Trp Asn Val Gly Asp Leu Val Met Trp Asp Asn ArgCys 245 250 255 260 gtg ttg cat aga ggt agg aga tat gat att tct gct agaagg gaa ctc 872 Val Leu His Arg Gly Arg Arg Tyr Asp Ile Ser Ala Arg ArgGlu Leu 265 270 275 aga agg gct act act ttg gat gac gct gtt gtt tagtgtgctggag 918 Arg Arg Ala Thr Thr Leu Asp Asp Ala Val Val 280 285

We claim:
 1. A method of improving the yield of a crop comprising:growing transgenic plants to produce a crop, the transgenic plants beingable to metabolize at least one synthetic auxin; applying a syntheticauxin to the plants at least once during their growth, the syntheticauxin being one that can be metabolized by the transgenic plants; andharvesting the crop.
 2. The method of claim 1 wherein the plants areannuals.
 3. The method of claim 1 wherein the plants are dicotyledonousplants.
 4. The method of claim 1 wherein the plants are tomato plants.5. The method of claim 1 wherein the plants are tobacco plants.
 6. Themethod of claim 1 wherein the plants are cotton plants.
 7. The method ofany one of claims 1-6 wherein the plants are able to metabolize at leastone phenoxy auxin.
 8. The method of claim 7 wherein the plants are ableto metabolize a phenoxy auxin selected from the group consisting of2,4-dichlorophenoxy acetic acid, 2,4-dichlorophenoxy butyric acid, andesters of either of them.
 9. The method of any one of claims 1-6 whereinthe auxin applied to the plants is a phenoxy auxin.
 10. The method ofclaim 9 wherein the auxin applied to the plants is a phenoxy auxinselected from the group consisting of 2,4-dichlorophenoxy acetic acid,2,4-dichlorophenoxy butyric acid, and esters of either of them.
 11. Themethod of claim 7 wherein the auxin applied to the plants is a phenoxyauxin.
 12. The method of claim 11 wherein the auxin applied to theplants is a phenoxy auxin selected from the group consisting of2,4-dichlorophenoxy acetic acid, 2,4-dichlorophenoxy butyric acid, andesters of either of them.
 13. The method of claim 8 wherein the auxinapplied to the plants is a phenoxy auxin selected from the groupconsisting of 2,4-dichlorophenoxy acetic acid, 2,4-dichlorophenoxybutyric acid, and esters of either of them.